Full text of "USPTO Patents Application 09544084"

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WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau

PCT

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 6 :

A23L 1730, A23D 7 AH) // A23C 9/152

(11) International Publication Number: WO 97/46118

(43) International Publication Date: It December 1997 (1 1.12.97)

(21) International Application Number: PCT/US97/04537

(22) International Filing Date: 19 March 1997 (19.03.97)

(30) Priority Data:

08/659,845

J June 1996 (07.06,96).

(71) Applicant: WISCONSIN ALUMNI RESEARCH FOUNDA-

TION (US/US]; P.O. Box 7365, Madison. WI 53707-7365
(US).

(72) Inventors: COOK. Mark, E.; 15 Kewaunee Court, Madison,

WI 53705 (US). PARIZA. Michael, W.; 7102 Valhalla
Trail, Madison, WI 53719 (US).

(81) Designated States: AL. AM, AT. AU. AZ, BA. BB, BG, BR,
BY, CA, CH. CN. CU. CZ. DE. DK. EE, ES. Fl. GB, GE,
GH f HU, IL. IS. JP t KE, KG, KP, KR, KZ, LC, LK, LR,
LS, LT, LU. LV, MD. MG, MK. MN. MW, MX, NO. NZ,
PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR. TT,
UA, UG, UZ, VN. YU, ARIPO patent (GH, KE, LS, MW,
SD, SZ, UG). Eurasian patent (AM, AZ. BY. KG, KZ. MD,
RU, TJ~, TM), European patent (AT, BE. CH, DE, DK. ES.
FI. FR, GB, GR, IE, IT. LU, MC, NL, PT, SE), OAPI patent
(BF. BJ. CF. CG. CI. CM, GA. GN. ML. MR, NE. SN, TD,
TG).

Published

With inUrnational search report.

(74) Agent: KRYSHAK. Thad; Quarles 8c Brady. 41 1 East Wiscon-
sin Avenue, Milwaukee. WI 53202-4497 (US).

(54) Title: DIETETIC FOODS CONTAINING CONJUGATED LINOLEIC ACIDS
(57) Abstract

A dietetic food which contains a safe and effective amount of conjugated linoleic acid (CLA).

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.

Albania

Spain

Lezcxho

Slovenia

Armenia

Poland

Lithuania

Slovakia

A asms

France

Luxembourg

Senegal

Anstralia

Gabon

Latvia

Swaziland

Azerhaijui

United Kingdom

Monaco

Chad

BocaU end Herzegovina

Georgia

Republic of Moldova

Togo

Barbados

Ohana

Madagascar

Tajtkssian

Belfium

Gumca

The former Yugoslav

Turkmen man

Burkina Paso

Greece

Republic of Macedonia

Turkey

Bulgaria

Hungary

Mali

Trinidad and Tobago

Beam

Ireland

Mongolia

Ukraine

Brazil

brael

Mauritania

Uganda

Belarus

Iceland

Malawi

United Suies of America

Canada

lUly

Mexico

Uzbek ulan

Central African Republic

Japan

Niger

Viet Nam

Congo

Kenya

Netherlands

YugoaUvii

Switzerland

Kyrgyutan

Norway

Zimbabwe

CCxe d'lvotre

Democratic People's

New Zealand

Cameroon

Republic of Korea

Poland

China

Republic of Korea

Portugal

Cute

Kaxakacaa

Romania

Cxech Republic

S&im Lucia

Russian Federation

GcfTwny

Liechtenstein

Sudan

Denmark

Sri Lanka

Estonia

Liberia

Singapore

WO 97/46118 PCT/US97/04537

-1-

DIETETIC FOODS CONTAINING CONJUGATED LINOLEIC ACIDS

Field of the Invention

The present invention generally relates to human
nutrition. More particularly, it relates to dietetic foods
for animals, especially humans.

Background of the Invention

Dietetic foods are synthetic foods specifically
formulated for people on restricted diets. Such foods, which
can contain natural foods as ingredients, can take the form of
either enteral compositions or parenteral compositions.

Enteral compositions are compositions for oral consumption
or tubal feeding intended to replace natural food products that
cause or aggravate allergies or other conditions in some
individuals. Some common examples of enteral compositions are
the baby formulae which do not contain milk proteins and
margarines intended for heart patients.

Parenteral compositions are compositions for intra-venous
administration to patients. Usually they are used with
patients who have difficulty with orally administered food.
Some common examples of parenteral compositions are solutions
of electrolytes, proteins, carbohydrates and fats.

We have discovered that it is advantageous for humans to
consume more conjugated linoleic acids (CLA) than are provided
in dietetic foods. It is especially important that humans who
are on restricted diets and consume only dietetic foods
receive conjugated linoleic acid because such diets can be
totally lacking in the CLA which can be found in some natural
foods which are consumed in a normal unrestricted diet.

In addition to being a good calorie source in dietetic
foods, CLA can be a valuable addition to dietetic foods
because we have found it to be effective in increasing body
protein or preventing the loss of body protein in a human,
increasing food efficiency in humans, and reducing body fat.
In addition, it appears to stimulate the immune system and to
increase the level of CD4 and CD8 cells.

SUBSTITUTE SHEET (RULE 26)

WO 97/46118 PCT/US97/04537

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Summarv of the Invention

It is an object of the present invention to disclose
dietetic foods which contain conjugated linoleic acids (CLA) .
We have discovered that dietetic foods which contain a
5 safe amount of an active form of a conjugated linoleic acid
(CLA), such as 9, 11 -octadecadienoic acid and 10,12-
- -^--^--^ ecac j^ enoic ac £^[~ an ester^heredf~,~~a non-toxic salt
thereof, and mixtures thereof, are a superior nutritional
product for animals on restricted diets.
10 It will be apparent to those skilled in the art that the

f orementioned* objects and other advantages may be achieved by
the practice of the present invention.

Description of the Preferred Embodiment

The dietetic foods of the present invention contain a
15 safe and effective amount of an active form of conjugated

linoleic acid (CLA) selected from a conjugated linoleic acid,
such as 9, 11-octadecadienoic acid and 10 , 12 -octadecadienoic
acid, an ester thereof, a non- toxic salt thereof, and mixtures
thereof. These dietetic foods will also contain one or more
20 proteins, electrolytes, carbohydrates, fats, vitamins or
minerals .

The amount of the CLA to be included in the dietetic food
will vary with the intended use of the food and whether the
dietetic food with CLA will be the sole source of nutrition.
25 However, since the CLA is a natural food ingredient and

relatively non- toxic, the amount which can be consumed is not
critical as long as it is enough to be effective and it is not
contraindicated in the patient's diet.

The practice of the present invention is further
30 illustrated by the examples which follow:

EXAMPLE 1

Synthesis of Conjugated Linoleic Acids (CLA)
From Linoleic Acid and Safflower Oil

35 Ethylene glycol (1000 g) and 500 g potassium hydroxide

(KOH) are put into a 4-neck round bottom flask (5000 ml). The
flask is equipped with a mechanical, stirrer, a thermometer a

WO 97/46118 PCT/US97/04537

-3-

reflux condenser, and a nitrogen inlet. (The nitrogen
introduced in first run through two oxygen traps) .
Nitrogen is bubbled into the ethylene glycol and KOH mixture
for 2 0 min and the temperature is then raised to 180° C.
5 1000 g of linoleic acid, corn oil, or saf flower oil then

is introduced into the flask. The mixture is heated at 180° C

under- an— inert— atmosphere -for— 2— 5— hours-

The reaction mixture is cooled to ambient conditions and
600 ml HCl is added to the mixture which is stirred for 15
10 min. The pH of the mixture is adjusted to pH 3 . Next, 200 ml
of water is added into the mixture and stirred for 5 min. The
mixture is transferred into a 5 L separatory funnel and
extracted three times with 500-ml portions of hexane .

The aqueous layer is drained and the combined hexane
15 solution extracted with four 250-ml portions of 5% NaCl
solution.

The hexane is washed three times with water. The hexane
is transferred to a flask and moisture in the hexane removed
with anhydrous sodium sulfate (Na 2 SO 4 ) . The hexane is

20 filtered through Whatman paper into a clean 1000 ml round
bottom flask and the hexane removed under vacuum with a
rotoevaporator to obtain the CLA. The CLA is stored in a dark
bottle under argon at -80° C until time of use.

This method can be modified so as to utilize only food-

25 grade reagents and solvents as listed in Food Chemicals Codex,
third edition, National Academy Press, 1981.

The active forms of CLA include, in addition to the free
acids, the non- toxic salts thereof, the active esters thereof,
such as triglycerides, and mixtures thereof.

30 The free conjugated linoleic acids (CLA) have been

previously isolated from fried meats and described as
anticarcinogens by Y. L. Ha, N. K. Grimm and M. W. Pariza, in
Carcinogenesis, Vol. 8, No. 12, pp. 1881-1887 (1987). Since
then, they have been found in some processed cheese products.

35 Y. L. Ha, N. K. Grimm and M. W. Pariza, in J". Agric. Food

Chem., Vol. 37, No. 1, pp. 75-81 (1987). The free acid forms
of the CLA may be prepared by isomerizing linoleic acid. The
terms "conjugated linoleic acids" and "CLA M as used herein are

WO 97/461 18 PCTrJS97/04537

-4-

intended to include 9, 11 -octadecadienoic acid, 10,12-
octadecadienoic acid; non-toxic salts thereof; esters thereof;
and mixtures thereof. The non-toxic salts of the free acids
may be made by reacting the free acids with a non- toxic base.
5 One method of synthesizing CLA is described in Example 1 .

However, CLA may also be prepared from linoleic acid by action

of— a— l-i^ol^-ie-ac-id--i-somerase--f-rom- a— irarml-ess— microorgaTii-sm,

such as the Rumen bacterium Butyrivibrio f ibrisolvens .
Harmless microorganisms in the intestinal tracts of rats and

10 other monogastric animals may also convert linoleic acid to
CLA (S. F. Chin, J. M. Storkson, W. Liu, K. Allbright and M.
W. Pariza, 1994, J. Nutr. 124; 694-701.

The CLA obtained by the practice of the described methods
of preparation contains one or more of the 9,11-

15 octadecadienoic acids and/or 10, 12 -octadecadienoic acids and
active isomers thereof. It may be free or bound chemically
through ester linkages. The CLA is heat stable and can be
used as is, or dried and powdered. The CLA is readily con-
verted into a non- toxic salt, such as the sodium or potas-sium

2 0 salt, by reacting chemically equivalent amounts of the free

acid with an alkali hydroxide at a pH of about 8 to 9 . CLA
also can be esterified to glycerol to form mono-, di - , and
triglycerides .

Theoretically, 8 possible geometric isomers of 9,11- and
25 10, 12 -octadecadienoic acid (c9, ell; c9,tll; t9,cll; t9,2tll;
cl0,cl2; cl0,tl2; tl0,cl2 and t!0,tl2) would form from the
isomerization of c9, cl2 -octadecadienoic acid. As a result of
the isomerization, only four isomers (c9,cll; c9,tll; tl0,cl2;
and cl0,cl2) would be expected. However, of the four isomers,
30 c9,tll- and tl0,c!2- isomers are predominantly produced during
the autoxidation or alkali-isomerization of c9,cl2-linoleic
acid due to the co-planar characteristics of 5 carbon atoms
around a conjugated double-bond and spatial conflict of the
resonance radical. The remaining two c,c- isomers are minor

3 5 contributors.

The relatively higher distribution of the t # t- isomers of
9,11- or 10, 12 -octadecadienoic acid apparently results from
the further stabilization of c9,tll- or t!0,cl2- geometric

WO 97/461 18 PCT/US97/04537

-5-

isomers, which is thermodynamically preferred, during an
extended processing time or long aging period. Additionally
the t,t-isomer of 9,11- or 10, 12 -octadecadienoic acid that was
predominantly formed during isomerization of linoleic acid
geometrical isomers (t9,tl2-, c9,tl2- and t9,cl2-
octadecadienoic acid) may influence the final ratio of the

"i"somers~or-l:he^iTiad-^IA— con tent—i-n-trhe-- samples t —

Linoleic acid geometrical isomers also influence the
distribution of minor contributors (c,c- isomers of 9,11- and

10.12- , t9,cll- and ell , tl2-octadecadienoic acids). The

11.13- isomer might be produced as a minor product from c9,cl2-
octadecadienoic acid or from its isomeric forms during
processing .

The exact amount of CLA tc be incorporated into a
dietetic food, of course, depends upon the intended use of the
food, the form of CLA employed, and route of administration.
It also can depend upon the isomer ratios. However, generally
the dietetic food will contain the equivalent of about 0.5 g
to about 1.0% g of CLA by weight of the dietetic food. The
CLA content also can be expressed as the amount of CLA based
on the total calories in the serving e.g. 0.03 to 3 gram CLA
per 100 calorie serving. Alternatively, the amount of CLA can
be expressed as a percentage of the lipid or fat in the food,
such as 0.3% to 100% of the food lipid, or as an amount of CLA
per gram of food lipid, such as 3 to 1000 mg CLA per gram of
lipid .

When the patient's sole source of food is the dietetic
food, the amount of CLA employed should be such that the
patient consuming the dietetic food will obtain from about 500
parts per million (ppm) to about 10,000 ppm of CLA in his
diet. If the dietetic food is not the sole source of food
higher or lower amounts of the dietetic food might need to be
consumed to reach these levels. However, the upper limit of
the amount to be employed is not critical because CLA is
relatively non-toxic and it is a normal constituent of the
human diet (including human breast milk) .

WO 97/461 18 PCT7US97/04537

-6-

The CLA to be incorporated into the dietetic food can be
in the form of the free acid, a salt thereof; an ester
thereof, such as a triglyceride; and any mixtures thereof.

Example 1

5 A liquid dietetic food for parenteral administration to

humans-eont-a-i-ns—emuis-i^-ied— f ^at-^ar-t-ie-l-es-of— about— 0-75-3^0—5 jxm

in diameter. In addition, the emulsions can contain Water for
Injection USP as a diluent, egg phosphatides (1-2%) as an
emulsifying agent and glycerin (2-3%) to adjust toxicity.
These emulsions can be infused intravenously to patients
requiring parenteral nutrition. Representative formulae of
the present invention would contain the same ingredients plus
0.5 mg/gm to 10 mg/gm of CLA or alternatively, 0.3% to 100%
CLA based on the food lipid or 0.03 gram to .3 gram per 100
15 calorie serving. For such parenteral foods the CLA usually
should be present in the form of the triglycerides.

Example 2

A milk protein- free, soy protein-based, baby formula is
prepared which contains CLA. Such a baby formula will contain
2 0 about 0.5 mg/gram to about 10 mg/gram of CLA or about 0.03

gram to 0 . 5 gram CLA per 100 calorie serving or 0.3% to 100%
CLA based on the lipid in formula.

One serving (100 calories) of a representative formula
can contain the following:
25 Protein 2.66 g

Fat 5.46 g

Carbohydrate 10.1 g

Water 133 g

CLA 0.3 g

30 Vitamins and Minerals (RDA amounts)

Example 3

A dietetic margarine of the present invention for use in
a heart-healthy diet is a semi-solid or solid vegetable oil-
based margarine which, in addition to the usual ingredients,
35 contains CLA. Such a margarine will contain about 0.25

WO 97/46118

-7-

PCT/US97/04537

mg/gram to about 10 mg/gm of CLA or about 0.03 gram to 0.5 gm
CLA per 100 calorie serving.

Example 4

A low residue liquid enteral dietetic product useful as a

5 high-protein, vitamin and mineral supplement contains added

CLA^ T he amount of CLA present can be about 0 . 05% to abo u t 5 %

by weight of CLA or about 0.3% to about 100% of the lipid

present or about 0.03 to 0.3 gram CLA per 100 calories.

One serving (140 calories) of a representative formula

10 can contain the following:

Protein (egg white solids) 7.5 g

Fat (CLA) 0.1 g

Carbohydrate (sucrose, 2 7.3g

hydrolyzed corn starch)

15 Water 1.9 g

Vitamins and Minerals (RDA amounts)

It will be readily apparent to those skilled in the art

that many dietetic foods, including those described in U.S.

Patent Nos. 4,282,265 and 5,470,839, can be improved by adding

20 CLA to the food or by replacing some of the fat in the food

with CLA.

It also will be readily apparent to those skilled in the
art that a number of modifications or changes may be made
without departing from the spirit and scope of the present
25 invention. Therefore, the invention is only to be limited by
the claims.

WO 97/46118

-8-

PCT/US97/04537

CLAIMS

1 . A dietetic food containing a member selected from
the class consisting of a conjugated linoleic acid, an ester
thereof, a non-toxic salt thereof, and mixtures thereof; said
member being present in amount of at least 3 mg per gram of
i-i-pi-d in— the— food-

2. A dietetic food of claim 1 in which the dietetic
food is a baby formula.

3. A dietetic food of claim 1 in which the dietetic
food is suitable for enteral administration.

4. A dietetic food of claim 1 in which the member is
present as an ester and dietetic food is suitable for
parenteral administration .

5. In a dietetic food, the improvement which comprises
incorporating in said food a safe amount of a member selected
from the class consisting of a conjugated linoleic acid, a
salt thereof, an ester thereof, and mixtures thereof.

6 . A dietetic food of claim 5 in which the amount of
the member is equivalent to at least about 3.0 mg per gram of
product lipid of the dietetic food.

7. A method of modifying a synthetic formulated
dietetic food which comprises incorporating in said food at
least about 3 . 0 mg per gram of CLA per gram of product lipid.

6. A dietetic food adapted for infant feeding as the
sole item of diet, said food comprising assimilable
carbohydrate, protein and fat, wherein the fat comprises at
least about 3 . 0 mg of CLA per gram of fat .

INTERNATIONAL SEARCH REPORT

Into nil Applic»bon No

PCT/US 97/04537

A. CLASSIFICATION OFSOBJECT MATTER , ,„„

IPC 6 A23L1/30 A23D7/0G //A23C9/152

Accordint to International Patent Classification (IPC) or to both national classification and IPC i

8. FIELDS SEARCHED

Minimum documentation searched (d aerification system followed by classification symbols)

IPC 6 A23L A23D A23C

Documentation searched other than minimum documentation to the exunt that such documents are inducted in the fields searched

Electronic data base consulted during the intemabonaJ jearch (name of data base and, where practical, search terms used)

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category *

Citation of document, with indication, where approprvaU, of the relevant passages

Relevant to daim No.

DMZ LEBENSMITTEL1NDUSTRIE UNO
MILCHWIRTSCHAFT,

vol. 116, no. 26, December 1995,

pages 1268-1272, XPG02035276

KAMMERLEHNER: "Linolsaure und konjugierte

Linolsauren - ihr Vorkorrmen im Milchfett,

ihre biologische Beudeutung w

see page 1270 - page 1271; tables 1,2

1,3.5

/--

j Further documents arc listed in the continuation of box C.

j Patent family members are listed

in annex.

* Special categories of dted documents

r *T* Later document published after the international filing date

A . fc . or priority date and not in conflict with the application but
A document defining the general state of the art which is not died to wtderstand the pnnop4c or theory underlying ihc

considered to be of particular relevance invention
* E * document but published on or after the international ^ document of particular relevance; the d aimed invention
aJtt cannot be considered novd or cannot be considered to
X" document which may throw doubti on priority daim(s) or involve in inventive step when the document if taken alone
which is ated to cxublish the publication date of another Y ' document of particular relevance; the daimed invention
atiboo or other tpeaal reason (as specified) cannot be eoradered to involve an invcnbve step when the
*O a document referring to an oral dtsdosurc, use. exhibition or document is combined with one or more other such docu-
other means menu, such combinabon being obvious to a person stalled
"P* document published prior to the intemabonal filing date but m the art.

later than the priority date daimed *A' document member of the same patent family

Dau of the

actual completion of the international search

Date of mailing of the international search report

15 July 1997

2 *». 07. 97

Name and mailing address of the ISA

European Patent Office, P.B. Silt Pattntlaan 2
NL • 2210 HV Rijswijt
Td. (-t Jl-70) 140-2040. Tx 31 631 cpo nl.
Fax: 31 -TO) 3an.]oi6

Authorized officer

Gac, G

i PCT7ISA/31* (i

Ami) i July ITU)

page 1 of 3

INTERNATIONAL SEARCH REPORT

Into. <ui Application No

PCT/US 97/04537

C(Condnuj

ition) DOCUMENTS CONSIDERED TO BE RELEVANT

Category *

Gut on of document, wiih indication, where ippropn.lc. of <hc rclcvinl puufts

Relevant to daim No.

v
A

lAiinuAt np r*n/\i\ rnunAP t tt au Aim aliaivctc

JOURNAL OF FOOD COMPOSITION AND ANALYMb,
„._vo1..5,.l January. 1992,

nanpC 1R5-1Q7 XPQflQ^ 751 1 1
pfllJC^ lOJ 13/ , ArUuVJ / JXJl

CHIN S F ET AL: "DIETARY SOURCES OF
CONJUGATED 0IEN0IC ISOMERS OF LINOLEIC
ACID, A NEWLY RECOGNIZED CLASS OF
ANTI CARCINOGENS"
see the whole document

1-3,8

— 5-7 - -
4

"Y
A

ruuu inert* ,

vol. 47, no. 3, 1993,

pages 257-261, XPOG2035277

5ANTHA ET AL.: "Conjugated linoleic acid

concentrations in processed cheese

contaning hydrogen donors, iron and

dairy-based additives"

see the whole document

1 3

J. FOOD SCI.,

vol. 60, no. 4, 1995,

pages O^D-DyO, /£U, AruUUO/DO*t!)

SANTHA ET AL. : Conjugated linoleic acid
concentrations in dairy products as
affected by processing and storage"
see the whole document

1,3

US 5 428 072 A (COOK) 27 January 1995

coo f ho uhnl o Hnrnmont

see tnt; wr icm e uoLumcnT.

1,5-7

.1 nMDV ^ri

vol. 78, no. 11, 1995,

pages 2358-2365. XPGG0676673

LIN ET AL. : "Survey of the conjugated

linoleic acid contents of dairy products"

see the whole document!

1 3
l , j

X
Y

ERNAHRUNG,

vol. 19, no. 6, 1995,

pages 265-270, XPGO0676658

SIEBER : "konjugierte Linolsauren in

Lebensmitteln : eine Ubersicht"

see the whole document

see page 266 - page 268

1.3
5

NUTRITION REPORTS INTERNATIONAL,

vol. 38, no. 5, November 1988,

pages 937-944, XP0066B7841

FOGERTY A C ET AL: "OCTADECA-9.11-DIEN0IC

ACID IN FOODSTUFFS AND IN THE L0P1DS OF

HUMAN BLOOD AND BREAST MILK"

see tables 1, 1 1

-/--

1.3-5

Form rCTyiXA/311 (a.rOnMt»M wt

•Met) (Jv* IW|

page 2 of 3

INTERNATIONAL SEARCH REPORT

Inter. "1 Application No

PCT/US 97/04537

^Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT

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